Streamlined methods for making liquid media

ABSTRACT

Provided herein are, inter alia, methods for preparing a liquid cell culture media that has lesser lot-to-lot analytical variation, increased performance, and has lesser metal ion concentrations compared to a liquid media prepared by traditional methods. Such liquid media may be used for culturing cells, including but not limited to, recombinant cells.

BACKGROUND

Liquid cell culture media manufacturing processes utilize large volumesof acids and bases to solubilize certain media components. Suchadditions into liquid formulations introduce lot-to-lot variability, maymodify or damage sensitive media components over time and may introducevariations in metal ion concentrations. There is a long felt need for areliable, efficient method for making liquid cell culture media, wheremetal ion concentrations can be controlled, and where there is reductionin the introduction of heavy metals, thereby reducing environmentalconcerns downstream.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the comparison of liquid media prepared by theconcentrate liquid method (table left) versus by the streamlined liquidmethod (table right). In one embodiment, the concentrate liquidtechnology took an average of 7 hrs production time whereas thestreamlined liquid took an average of 3-4 hrs production time.

FIG. 2 Concentrate liquid method uses high quantities of acids and basesduring preparation of the liquid stock components. The table shows that,in an exemplary liquid media prepared by concentrate technology, acids &bases account for 1.88% of the final volume. Here, concentratetechnology used 18.804 mL/L of acid/base. The streamlined liquid methodcompletely eliminated the use of 12N concentrated acid and significantlyreduced the use of 5N acid and 5N base, thereby reducing theintroduction of metal contaminants into the final formulation.

FIG. 3 depicts various metal concentrations (contaminants) measured inacid (5N HCl) and base (5N NaOH). Four lots of 5N HCl and 5N NaOH wereanalyzed by ICP-MS for metal contaminants Present concentrate liquidtechnology uses high volumes of acids and bases, which contributes tothe variability in metal contaminant concentrations in the final liquidmedium. (A) depicts amount of metals present in higher concentrationscompared to metals that are less than 30 μg/L shown in (B). Thestreamlined liquid method eliminates the use of 12N concentrated acid,and significantly reduces the use of 5N acid and 5N base, therebyreducing the concentration and variability associated with metalcontaminants.

FIG. 4 shows the comparison of metal levels in media prepared by theconcentrate liquid method versus by the streamlined liquid method. Mediaprepared by both these methods were analyzed by ICP-MS. The metalconcentrations were normalized such that 100% represents the metalspresent in the ‘concentrate’ method (the control). (A) The streamlinedmethod for preparing liquid CDCHO™ medium (from Life Technologies,Corp., Carlsbad, Calif.) resulted in lower metal concentrations of:chromium, copper, manganese and nickel compared to when prepared by the‘concentrate’ method (control). (B) The streamlined method for preparingany liquid 293 medium (from Life Technologies, Corp., Carlsbad, Calif.)resulted in lower metal concentrations of: chromium, iron, manganese andnickel compared to when prepared by the ‘concentrate’ method (control).

FIG. 5 depicts the comparison of cell growth measured by viable celldensities (VCD) of (A) CHO-S cells which showed improved growth of 147%in the streamlined liquid media, compared to the concentrate liquidcontrol. (B) 293 cells had similar growth performance in the StreamlinedLiquid media (105%) when compared to the Concentrate Liquid control.

FIG. 6 shows the IgG titer performance comparison in liquid CDCHO™ mediaprepared by concentrate liquid method versus by the streamlined liquidmethod. In media prepared by the ‘streamlined’ method, CHO-S cellsdemonstrated increased protein productivity of about 124% when comparedto protein productivity in liquid medium prepared by the traditional‘concentrate’ control method.

The present disclosure provides a method of making a liquid mediumcomprising:

-   -   i) grouping media components into groups based on their pH,        solubility and concentration,    -   ii) converting an amino acid's free base form or salt form to        target a particular pH according to the components in the        formulation;    -   iii) performing addition of the groups in an order that permits        optimal solubilization of said group;        -   wherein the resulting liquid medium requires lesser acid            and/or base addition to solubilize the groups compared to a            medium that is not prepared using steps i) ii) and iii)            above (steps i), ii) and iii) form the streamlined method).

The resultant liquid medium using this method: (a) required lesser acidand/or base additions to solubilize the components; and/or, (b) hadreduced metal contaminants from acid and/or base additions; and/or, (c)had less lot-to-lot variability in metals, and, (d) required lesserformulation time since lesser solutions needed to be prepared comparedto a liquid medium prepared by another method not using the steps i),ii) and iii) described above for liquid medium preparation.

The liquid medium prepared by the method described above had severaladvantages: for instance, in one embodiment, the lot-to-lot variabilitydue to metals in the liquid was reduced to less than up to 10%. In otherembodiments, the lot-to-lot variability of total metal concentrationswas reduced to less than about 0.001%, less than about 0.01%, less thanabout 0.1%, less than about 1%, less than about 1-2%, less than about1-3%, less than about 1-4%, less than about 1-5%, less than about 1-6%,less than about 1-10%, less than about 1-20%, less than about 1-30%,less than about 1-40%, less than about 1-50%, less than about 1-60%,less than about 1-70%, %, less than about 1-80%, or less than about1-90%. In other embodiments, the lot-to-lot variability for any metalconcentration was less than about 0.001%, less than about 0.01%, lessthan about 0.1%, less than about 1%, less than about 2%, less than about3%, less than about 4%, less than about 5%, less than about 6%, lessthan about 10%, less than about 15%, less than about 20%, less thanabout 30%, less than about 40%, less than about 50%, less than about0.001-1%, less than about 0.001-5%, less than about 0.001-10%, less thanabout 1-10%, less than about 10-20%, less than about 20-30%, less thanabout 30-40%, less than about 40-50%, less than about 50-100%, less thanabout 50-90%, less than about 50-80%, less than about 50-70%, less thanabout 50-60%.

Another advantage in using the streamlined method is that the volume ofacid and/or base addition to the liquid is reduced. For instance, insome embodiments, the reduction in the volume of acid and/or base isabout 10-40 fold. In a particular embodiment, the reduction in thevolume of acid and/or base is about 22 fold. In a certain embodiment,the use of 12N concentrated acid is eliminated in the streamlinedmethod.

In one aspect, the reduction of the one or more contaminant metal in theliquid medium was about 0.0001% to 100% compared to liquid mediumprepared by any other method, for e.g., the concentrate method. In otheraspects, the reduction of the one or more contaminant metal is by about0.0001% to 0.001%, about 0.0001% to 0.01%, about 0.0001% to 0.1%, about0.0001% to 1%, about 0.0001% to 2%, about 0.0001% to 3%, about 0.0001%to 4%, about 0.0001% to 5%, about 0.0001% to 10%, about 1%-5%, about1%-10%, about 1%-15%, about 1%-20%, about 1%-25%, about 1%-35%, about10-20%, about 10-30%, about 10-40%, about 10-50%, about 10-60%, about10-70%, about 10-80%, about 10-90%, about 10-100% compared to liquidmedium prepared by any other method, for e.g., the concentrate method ofpreparing liquid media.

In one aspect, the contaminant metals that are reduced in the liquidmedium are selected from the group consisting of: Cr, Fe, Mg, Cu, Mn,Ni, Zn, Mo, Al and Ca. In another aspect, the % metal contamination isreduced by: about 40-100% for Cr, about 0.01-20% for Fe, about 0.1-15%for Cu, about 0.25-60% for Mn, about 5-100% for Ni, about 5% for Zn, andabout 15% for Mo compared to liquid medium prepared by any other method,for e.g., the concentrate method. In a particular aspect, the liquidcell culture medium has lower contaminant metal levels than a liquidmedium prepared by another method.

The present disclosure also provides a liquid cell culture medium thatis serum-free, a liquid cell culture medium that is protein-free, orboth.

The present disclosure also provides methods for culturing a cell,comprising contacting the cell with the liquid cell culture mediumprepared using the methods described above. In a preferred embodiment,the cells are selected from the group consisting of: primary epithelialcells (e.g., keratinocytes, cervical epithelial cells, bronchialepithelial cells, tracheal epithelial cells, kidney epithelial cells andretinal epithelial cells), established cell lines and their strains,recombinantly engineered cells, diploid cells, hybriomas, Chinesehamster ovary (CHO), HEK293 cells, TRG-2 cells, IMR-33 cells, Don cells,GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells,Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells,CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells,HSDM₁C₃ cells, KLN₂O₅ cells, McCoy cells, Mouse L cells, Strain 2071(Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells,NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indianmuntjac cells, SIRC cells, C_(II) cells, and Jensen cells, Sp2/0, NS0,NS1 cells or derivatives thereof and iPSC cells.

The present disclosure further provides the cell that can eitherexpress: a recombinant product, a protein or a recombinant protein, afusion product or protein, an antibody or an antibody fragment, a fusionor modified antibody, a virus or a viral product or component, a VLP(viral like particle), a cell, a nucleic acid, a lipid, a hormone, asteroid, or a glycoprotein.

The present disclosure further provides methods for making a biologicalproduct in a liquid medium prepared according to claim 3 13-18, whereinthe biological product is a recombinant product, a protein or arecombinant protein, a fusion product or protein, an antibody or anantibody fragment, a fusion or modified antibody, a virus or a viralproduct or component, a VLP (viral like particle), a cell, a nucleicacid, a lipid, a hormone, a steroid, or a glycoprotein.

The present disclosure provides a liquid cell culture medium comprisingreduced metal contaminants and/or showing reduced lot-to-lot variabilityin metals compared to a liquid medium prepared using another methodwherein it uses acid and/or base additions to solubilize its components.

The present disclosure also provides a liquid cell culture mediumprepared according to the methods described above (the streamlinedmethod), wherein the liquid medium has less contaminant metals comparedto liquid medium prepared by any other method, for e.g., the concentratemethod of preparing liquid media.

The present disclosure also provides a kit or a combination comprising:(i) a liquid cell culture medium prepared according to the method ofclaims 1-12, (ii) cells, and optionally, (iii) a cell culture supplementor additive.

Description 1. Cell Culture Medium

A cell culture medium is composed of a number of ingredients and theseingredients vary from one culture medium to another. A cell culturemedium may be a complete formulation, i.e., a cell culture medium thatrequires no supplementation to culture cells, or, may be an incompleteformulation, i.e., a cell culture medium may require supplementation ormay be used as a supplement to supplement an incomplete formulation, or,in the case of a complete formulation, may improve cell cultureperformance or culture results (titers).

Generally a cell culture medium may have solutes dissolved in solvent.The solutes provide an osmotic force to balance the osmotic pressureacross the cell membrane (or wall). Additionally, the solutes mayprovide nutrients for the cell. Some nutrients may be chemical fuel forcellular operations; some nutrients may be raw materials for the cell touse in anabolism; some nutrients may be machinery, such as enzymes orcarriers that facilitate cellular metabolism; some nutrients may bebinding agents that bind and buffer ingredients for cell use or thatbind or sequester deleterious cell products.

Depending on the cell and the intended use of the cell, the ingredientsof the cell culture medium can be determined at concentrations tobalance/optimize cell culture performance. Performance may be measuredin accordance with a one or more desired characteristics, for example,cell number, cell mass, cell density, 02 consumption, consumption of aculture ingredient, such as glucose or a nucleotide, production of abiomolecule, secretion of a biomolecule, formation of a waste product orby product, e.g., a metabolite, activity on an indicator or signalmolecule, etc. Each or a selection of the ingredients may thuspreferably optimized to a working concentration for the intendedpurpose.

Culture media or feed supplements of the invention may be available in adry format that requires only addition of a solvent such as water. Dryformats include, but are not limited to, dry powder format (DPM),agglomerated (AGT™) format, advanced powder media (APM), or othersuitable dry formats, and have been described in other patents.Preferably, once water is added, dissolution should occur quickly andthe resultant liquid can be filtered and added directly to the cellswithout any pH adjustment. The reconstituted medium or concentratedsupplement may be prepared in variable bulk quantities and is amenableto sterilization, particularly by ionizing or ultraviolet irradiation.Media (dry and liquid) generally comprise the following:

a. Carbohydrates

Cell culture medium ingredients typically include a carbohydrate, aminoacids, salts, trace elements, and vitamins. For mammalian cells, themain carbohydrate used in cell culture media is glucose, routinelysupplemented at 5 to 25 nM. See Cell Culture Technology forPharmaceutical and Cell-Based Therapies, 51 (Sadettin Ozturk andWei-Shou Hu eds., Taylor and Francis Group 2006). In addition toglucose, any hexose like galactose, fructose, or mannose or acombination of these may be used. In addition, mammalian cells can alsouse glutamine as a major energy source. Glutamine is often included athigher concentrations than other amino acids (2-8 mM). However, as notedabove, glutamine can spontaneously break down to form ammonia andcertain cell lines produce ammonia faster, which is toxic. Therefore,glutamate and glutamine dipeptides have been used as substitutes forglutamine to reduce the build-up of toxic ammonia in the cell culturemedium.

b. Amino Acids

Amino acids are important in cell culture medium for maintaining themetabolic function of the cultured cells. Cell culture medium typicallyincludes the essential amino acids (i.e., those amino acids that arenormally not synthesized in vivo by mammals) as well as certainnon-essential amino acids. A non-essential amino acid is typicallyincluded in the cell culture medium if the cell line is not capable ofsynthesizing the amino acid or if the cell line cannot producesufficient quantities of the amino acid to support maximal growth.Exemplary amino acids include L-alanine, L-arginine, L-asparagine,L-aspartic acid, L-cysteine, L-glutamic acid, L-glutamine, glycine,L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine,L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine,L-tryptophan, L-tyrosine, and L-valine. The methods in this disclosureare directed to classifying amino acids, based on their properties asshown, for example, in Table 1. Such classifications are well known.However this disclosure aims at grouping and matching the amino acids inpH and concentration, and matching their addition so as to reduce oreliminate the addition of acids or base to achieve a desired pH, withoutcompromising cell culture growth and titer performance from previouslyused methods using concentrate technology. Media concentrate technologyhas been described, for instance, in US patent application 5474931,whose disclosure is hereby incorporated by reference. An exemplaryclassification of amino acids is shown below in Table 1, where aminoacids are grouped based on their acidic, basic or neutral properties.Titration of the amino acid groups, and matching of the groups based onpH and concentration while preparing the liquid media can achieve adesired pH without addition of as much acid and/or base described in5474931, and as shown in the example below.

TABLE 1 Properties Amino acids Basic Arg, Lys, His Acidic Asp, GluNeutral (non polar) Ala, Asn, Gln, Gly, Ile, Leu, Met, Phe, Pro, ValNeutral (slightly polar) Cys, Trp, Neutral (polar) Ser, Thr, Tyr,

c. Salts

Salts are added to cell culture medium to maintain isotonic conditionsand prevent osmotic imbalances. The osmolality of standard mammaliancell culture medium is about 300 mOsm/kg, although many cell lines cantolerate an approximately 10% variation of this value. The osmolality ofsome insect cell cultures tend to be higher than 300 mOsm/kg, and thismay be 0.5%, 1%, 2 to 5%, 5-10%, 10-15%, 15-20%, 20-25%, 25-30% higherthan 300 mOsm/kg. The most commonly used salts in cell culture mediuminclude Nat, K⁺, Mg²⁺, Ca²⁺, Cl⁻, SO₄ ²⁻, PO₄ ³⁻, and HCO₃ ⁻ (e.g.,CaCl₂), KCl, NaCl, NaHCO₃, Na₂HPO₄). Therefore, the desired osmolalityfor a cell culture medium for cultivation of a particular cell type mayalso be determined empirically by one of ordinary skill in the art,using art-known methods.

d. Inorganic Elements

Other inorganic elements that are present in serum in trace amounts canbe included in cell culture medium, as described in US 2005/0287666,which is hereby incorporated by reference in its entirety. They includeMn, Cu, Zn, Mo, Va, Se, Fe, Ca, Mg, Si, and Ni. Other inorganic elementsthat have been added to cell culture medium, although not as frequently,include Al, Ag, Ba, Br, Cd, Co, Cr, F, Ge, J, Rb, and Zr. Many of theseelements are involved in enzymatic activity. They may be provided in theform of salts such as CaCl₂, Fe(NO₃)₃, MgCl₂, MgSO₄, MnCl₂, NaCl,NaHCO₃, Na₂HPO₄, and ions of the trace elements, such as, selenium,vanadium and zinc. These trace elements may be provided in a variety offorms, preferably in the form of salts such as Na₂SeO₃, NH₄VO₃, etc.These inorganic salts and trace elements may be obtained commercially,for example from Sigma (Saint Louis, Mo.).

e. Vitamins

Vitamins are typically used by cells as cofactors. The vitaminrequirements of each cell line vary greatly, although generally extravitamins are needed if the cell culture medium contains little or noserum or if the cells are grown at high density. Exemplary vitaminsinclude biotin, choline chloride, folic acid, i-inositol, nicotinamide,D-Ca⁺⁺-pantothenate, pyridoxal, riboflavin, thiamine, pyridoxine,niacinamide, A, B₆, B₁₂, C, D₃, E, K, and p-aminobenzoic acid (PABA).

f. Optional: Serum or Recombinant Serum Proteins

Serum, the supernatant of clotted blood, can be used in cell culturemedium to provide components that promote cell growth and/orproductivity. These serum components include attachment factors,micronutrients (e.g., trace elements), growth factors (e.g., hormones,proteases), and protective elements (e.g., antitoxins, antioxidants,antiproteases). Serum is available from a variety of animal sourcesincluding bovine or equine. When included in cell culture medium, serumis typically added at a concentration of 5-10%. Certain cell culturemedia are serum free.

g. Growth Factors

To promote cell growth in the absence or serum or in serum reducedmedia, one or more of the following polypeptides can be added to a cellculture medium: for example, fibroblast growth factor (FGF), includingacidic FGF and basic FGF, insulin, insulin-like growth factor (IGF),epithelial growth factor (EGF), nerve growth factor (NGF),platelet-derived growth factor (PDGF), and transforming growth factor(TGF), including TGFα and TGFβ, any cytokine, such as interleukins 1, 2,6, granulocyte stimulating factor, Leukocyte inhibitory factor (LIF),etc.

In certain embodiments, the cell culture medium does not contain agrowth factor. In protein-free media, insulin may be replaced with zincor a zinc containing compound, as described in WO 98/08934, which ishereby incorporated by reference in its entirety.

h. Lipids

One or more lipids can also be added to a cell culture medium. Serumtypically contains lipids, such as fatty acids (e.g., linoleic acid,linolenic acid, arachidonic acid, palmitoleic acid, oleic acid,polyenoic acid, and/or fatty acids of 12, 14, 16, 18, 20, or 24 carbonatoms, each carbon atom branched or unbranched), phospholipids, lecithin(phophatidylcholine), and cholesterol. Alternatively, one or more ofthese lipids can be included as supplements in serum-free media.Phosphatidic acid and lysophosphatidic acid stimulate the growth ofcertain anchorage-dependent cells, such as MDCK, mouse epithelial, andother kidney cell lines, while phosphatidylcholine,phosphatidylethanolamine, and phosphatidylinositol stimulate the growthof human fibroblasts in serum-free media. Ethanolamine and cholesterolhave also been shown to promote the growth of certain cell lines. Incertain embodiment, the cell culture medium does not contain a lipid.

i. Optional: Carrier Proteins

One or more carrier proteins, such as bovine serum albumin (BSA) ortransferrin, can also be added to the cell culture medium. Carrierproteins can help in the transport of certain nutrients or traceelements. BSA is typically used as a carrier of lipids, such as linoleicand oleic acids, which are insoluble in aqueous solution. In addition,BSA can also serve as a carrier for certain metals, such as Fe, Cu, andNi. In protein-free formulations, non-animal derived substitutes forBSA, such as cyclodextrin, can be used as lipid carriers. Transferrin isinvolved in transporting iron across cell membranes. In certain cases,human serum albumin may be necessary for the cultivation of cells (for.e.g., such as in xeno-free (XF) culture) desirable for productsgenerated for downstream therapeutic use. In other instances,recombinant human serum albumin may be used in the cell culture mediumfor the cultivation of cells. In particular cases, the recombinant humanserum albumin may be derived from plant, algal or fungal sources such asrice, corn, potato, wheat, even yeast, etc. to provide for animal-originfree (AOF) culturing of cells. In protein-free formulations, transferrincan be replaced by ferric and/or ferrous salts, as described in WO98/08934, which is hereby incorporated by reference in its entirety, ora hydroxypyridine derivative, as described in US 2007/0254358, which ishereby incorporated by reference in its entirety. Additionally, inprotein-free formulations, insulin can be replaced by zinc, vanadium orother suitable divalent salts.

j. Optional: Attachments Proteins

One or more attachment proteins, such as fibronectin, laminin, andpronectin, can also be added to a cell culture medium to help promotethe attachment of anchorage-dependent cells to a substrate.

k. Buffering Agent

The cell culture medium can optionally include one or more bufferingagents. Suitable buffering agents include, but are not limited to,N-[2-hydroxyethyl]-piperazine-N′-[2-ethanesulfonic acid] (HEPES), MOPS,MES, phosphate, bicarbonate and other buffering agents suitable for usein cell culture applications. A suitable buffering agent is one thatprovides buffering capacity without substantial cytotoxicity to thecells cultured. The selection of suitable buffering agents is within theambit of ordinary skill in the art of cell culture.

1. Polyanionic or Polycationic Compounds

Polyanionic or polycationic compounds can prevent the cells fromclumping and promote growth of the cells in suspension. See WO 98/08934,which is hereby incorporated by reference in its entirety. Exemplarypolyanionic compounds include polysulfonated or polysulfated compound,such as, heparin, dextran sulfate, heparan sulfate, dermatan sulfate,chondroitin sulfate, pentosan polysulfate, a proteoglycan or the like.

In addition, the cell culture medium may comprise one or moreingredients described here. In one embodiment, the cell culture mediummay optionally comprise one or more of the following ingredients:ethanolamine, D-glucose, HEPES, insulin, a cytokine (e.g., IL-6),heparin, dextran sulfate, linoleic acid, lipoic acid, phenol red,PLURONIC® F68, putrescine, sodium pyruvate, transferrin, L-alanine,L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-glutamic acid,L-glutamine, glycine, L-histidine, L-isoleucine, L-leucine, L-lysine,L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine,L-tryptophan, L-tyrosine, L-valine, biotin, choline chloride,D-Ca⁺⁺-pantothenate, folic acid, i-inositol, niacinamide, pyridoxine,riboflavin, thiamine, vitamin B₁₂, one or more calcium salts, Fe(NO₃)₃,KCl, one or more magnesium salts, one or more manganese salts, NaCl,NaHCO₃, Na₂HPO₄, one or more selenium salts, one or more vanadium saltsand one or more zinc salts.

The liquid media described can be a 1× formulation, or can beconcentrated as anything greater than a 1× formulation, for example, asa 2×, 5×, 10×, 20×, 50×, 500×, or 1000× medium formulation as thesolubility of the individual components allow. If the medium ingredientsare prepared as concentrated stock solutions by the concentrate method,an appropriate (sufficient) amount of each concentrate stock is combinedwith a diluent to produce a 1× medium formulation. Typically, thediluent used is water but other solutions including aqueous buffers,aqueous saline solution, or other aqueous solutions may be used. The 1×solution is prepared directly by the streamlined method, and isdescribed further below and in the Examples. There is no need for thepreparation of concentrated stock solutions in the streamlined method.

Methods for Preparing Liquid Media

Traditional Concentrate Method

A liquid 1× can be prepared using the ‘concentrate’ technology, asdetailed in U.S. Pat. No. 5,474,931. In this ‘concentrate’ method,components or ingredients (as seen in Tables 2-5) can be prepared andstored in solution. Preferably, ingredients are grouped in concentratedsolutions and stored. For example, a grouping may be the groups of aminoacid ingredients shown in Tables 1. An exemplary grouping of theingredients of Table 2 may be: i) acid soluble amino acids in one group,ii) certain vitamins and salts grouped together based on theirreactivities, concentrations and compatibility; iii) trace metalsgrouped according to their solubility properties; iv) pluronic, glucose,buffers, other vitamins and salts grouped together based on theirreactivities, concentrations and compatibility.

Stock solutions of the grouped ingredients can be made as concentratedstocks. For example, it is possible to make 10× to 100× chemical stocksolutions, which can be stored as liquids or frozen in the appropriatealiquot sizes for later use.

Stock solutions offer a number of advantages. For example, a higherfinal concentration of a given ingredient can be used in the 1× medium.In addition, some ingredients are more stable when stored in aconcentrated stock solution. Moreover, less storage volume is requiredfor a concentrated stock solution than is required for a 1×medium. SeeU.S. Pat. No. 5,474,931. For exemplary methods on how to prepare stocksolutions using the ‘concentrate’ method or for an exemplary grouping ofacid soluble I, acid soluble II, salt I, salt II, see U.S. Pat. No.8,198,084 (columns 25, 26, 27, 28-29, and Table 2 of U.S. Pat. No.8,198,084 is hereby incorporated by reference in its entirety).

Streamlined Method

On the other hand, a liquid prepared using the ‘streamlined’ technologydoes the following:

-   -   i) Grouping: Components are grouped by type, pH, concentration        and solubility, for e.g. alkaline amino acids, like Arg & Lys        are grouped together; or amino acids are grouped into multiple        sub-groups according to pH with acidic, basic and neutral        groupings, and with the order of addition optimized based on        theoretical solubilities, pH and bench top testing. An exemplary        order of amino group addition to the formulation tank would be        as follows: 1) acidic, 2) basic and 3) neutral;    -   ii) Converting an amino acid's free base form or salt form to        target a particular pH according to the components in the        formulation; and/or, converting tyrosine and cystine from free        base to disodium salt forms, as appropriate for the formulation;        and/or, converting phosphate forms from dibasic sodium phosphate        to a combination of monobasic and dibasic forms to achieve a        desired final target pH;    -   iii) Reducing addition of acid and/or base: The largest source        of inconsistent trace metal contaminants from raw materials        comes from acids and bases, for e.g., 5N HCl and NaOH, 12 N HCl,        etc. used for adjusting pH and for solubilization of components.        Since the proposed streamlined process limits, and in many cases        eliminates the need for large volume additions of acid and base,        there is less lot to lot variation due to the contribution of        metal element concentrations in acids and bases (see FIG. 3);    -   iv) Lesser side solutions prepared: Part of the streamlined        approach is to minimize the number of solutions (or stock        solutions, also referred to as side solutions) and        pre-solubilization steps. Hence, dry/wet components may be used        directly and not “prepared as solutions”, unlike the preparation        of stock solutions in the ‘concentrate technology’. Hence,        components present in large volumes, such as acid soluble        groupings I & II, salts I & II which were prepared into        solutions are instead, added directly in their native forms        (that is, if the raw material is a dry powder, the dry powder is        added directly to the formulation preparation tank, or if the        raw material is a liquid, the liquid is added directly) into the        formulation tank in the streamlined approach.    -   v) Pooling trace solutions: Traditionally in the ‘concentrate’        method—multiple trace element solutions were prepared as stock        solutions. Instead, in streamlined, the multiple trace element        solutions are combined together into one complete “trace element        solution” leading to one, main tank addition instead of up to        eight additions. Typically, high concentration components like        amino acids and salts are added first before addition of the        lower concentration, more sensitive components. A general order        for addition of components can be as shown in FIG. 1:        ‘streamlined liquid table’ to the right, where, in one        embodiment, ingredients are added in the order shown from top to        bottom of the table.

Therefore, liquids prepared by the streamlined method:

-   -   (a) require lesser acid and/or base additions to solubilize the        components; and/or,    -   (b) have reduced metal contaminants from acid and/or base        additions; and/or,    -   (c) have lesser lot-to-lot variability in metals,    -   (d) requires lesser formulation time since lesser (side)        solutions need to be prepared and due to optimized groupings of        components, compared to a liquid medium prepared by say the        concentrate method. More time is needed to fully solubilize        components with wide ranges of pHs and solubilities of        concentrated method.

Generally, there is lot-to-lot variability at least in the metal ionconcentrations due to the addition of acids and/or bases for thesolubilization of components. Due to the reduction in the addition ofthe volume of acids and bases, and due to the fact that the streamlinemethod totally eliminates 12N concentrated acid in the protocol, thefold reduction in acid/base additions streamlined are about 1-10 fold,1-20 fold, 1-30 fold, or 1-40 fold, depending on the formulation. In oneembodiment, the reduction of acid/base volume was about 22 fold.

The lot-to-lot variability of metals can be measured by a variety ofanalytical methods, including but not limited to ICP-MS, MS/MS, etc.Either total metal ion concentrations, or specific metal ionconcentrations can be measured using suitable analytical methods and themetal ion differences are compared between lots. In a certainembodiment, the lot-to-lot variability of total metal concentration inthe streamlined method was reduced by up to 10%. In other embodiments,the total metal lot-to-lot variability was reduced by: about 0.001%,about 0.01%, about 0.1%, about 1%, about 1-2%, about 1-3%, about 1-4%,about 1-5%, about 1-6%, about 1-10%, about 1-20%, about 1-30%, about1-40%, about 1-50%, about 1-60%, about 1-70%, %, about 1-80%, or about1-90%.

In other embodiments, individual metal concentration can be measuredbetween various liquid lots. The metals that are measured are selectedfrom the group consisting of: Cr, Fe, Mg, Cu, Mn, Ni, Zn, Mo, Al and Ca.There may be reduction of any metal between the lots of streamlinedmedia as follows: less than about 0.001%, less than about 0.01%, lessthan about 0.1%, less than about 1%, less than about 2%, less than about3%, less than about 4%, less than about 5%, less than about 6%, lessthan about 10%, less than about 15%, less than about 20%, less thanabout 30%, less than about 40%, less than about 50%, less than about0.001-1%, less than about 0.001-5%, less than about 0.001-10%, less thanabout 1-10%, less than about 10-20%, less than about 20-30%, less thanabout 30-40%, less than about 40-50%, less than about 50-100%, less thanabout 50-90%, less than about 50-80%, less than about 50-70%, less thanabout 50-60%. The reduction of the one or more contaminant metal may becompared to liquid medium prepared by any other method, for e.g., theconcentrate method of preparing liquid media. In a certain embodiment,the reduction was about 0.0001% to 100%. Or the reduction of the one ormore contaminant metal is by about 0.0001% to 0.001%, about 0.0001% to0.01%, about 0.0001% to 0.1%, about 0.0001% to 1%, about 0.0001% to 2%,about 0.0001% to 3%, about 0.0001% to 4%, about 0.0001% to 5%, about0.0001% to 10%, about 1%-5%, about 1%-10%, about 1%-15%, about 1%-20%,about 1%-25%, about 1%-35%, about 10-20%, about 10-30%, about 10-40%,about 10-50%, about 10-60%, about 10-70%, about 10-80%, about 10-90%,about 10-100% in the resultant liquid prepared by the streamlined methodcompared to liquid medium prepared by any other method, for e.g., theconcentrate method.

In certain embodiments, the individual % metal contamination is reducedby: about 40-100% for Cr, about 0.01-20% for Fe, about 0.1-15% for Cu,about 0.25-60% for Mn, about 5-100% for Ni, about 5% for Zn, and about15% for Mo compared to liquid medium prepared by any other method, fore.g., the concentrate method.

The liquid medium prepared by the streamlined method maybe serum-free orprotein-free or both, low serum, low protein, or a combination thereof.The liquid medium may be used for culturing a cell.

2. Serum-Free Medium

Potential problems associated with serum, including batch to batchvariation, high protein content, risk of contaminants (e.g., viruses,mycoplasma, prions), limited availability, and high cost, have driventhe production of serum-free media. Furthermore, improved levels ofrecombinant protein expression can be obtained from cells grown inserum-free medium, relative to the level of expression seen in cellsgrown in medium supplemented with serum (Battista, P. J. et al., Am.Biotech Lab. 12: 64-68 (1994)).

In these serum-free media, serum can be replaced with a defined hormone,or hormone cocktails, such as HITES or ITES, which containhydrocortisone, insulin, transferrin, ethanolamine, and selenite.Alternatively, the serum-free media can contain growth factor extractsfrom endocrine glands, such as epidermal or fibroblast growth factors.Serum-free media can also contain other components as a substitute forserum, including purified proteins (animal or recombinant), peptones,amino acids, inorganic salts, and animal or plant hydrolysates (orfractions thereof).

Serum-free media may be chemically defined or undefined. In chemicallydefined media, the identity of the components and their amounts areknown, whereas the opposite is true for chemically undefined media.Chemically defined media, therefore, are designed, in part, to reducethe risk of contaminants and to reduce batch to batch variation.Chemically defined supplements that can be added to cell culture mediainclude growth factors, hormones, carrier proteins, and/or attachmentfactors. In a preferred embodiment, the basal medium used with the mediaor feeds comprising small peptides is a chemically-defined medium. Inanother preferred embodiment, the concentrated cell culture media orconcentrated feed of the invention comprising small peptides is also achemically-defined composition. In yet another preferred embodiment, theconcentrated feed or medium of the invention comprising small peptidesincluding cysteine and tyrosine is a single part feed and ischemically-defined. In another aspect, all the above compositionscomprising small peptides including cysteine and tyrosine are auto-pHand auto-osmolality balanced. In yet another aspect, all the abovecompositions comprising small peptides including cysteine and tyrosineare stoichiometrically balanced.

3. Protein-Free Media

Serum-free media contains reduced amounts of protein as compared to cellculture media containing serum. However, serum-free media may stillcontain one or more of a variety of animal-derived components, includingalbumin, fetuin, various hormones and other proteins. The presence ofproteins makes purification of recombinant protein difficult,time-consuming, and expensive and can also lead to reduced productyields and/or purity. Thus, in one embodiment, the cell culture mediumis protein free.

Protein-free media can be obtained by methods known in the art, such asby removing any remaining proteins from serum-free media. While theremoval of such proteins from the cell culture media can impair themedia's ability to support cell growth, other components can be added tothe media to mitigate the effect of removing the proteins from themedia. For example, as discussed above, cyclodextrin can replace BSA andiron salts or a hydroxypyridine derivative can replace transferrin. Inother cases, animal tissue or plant hydrolysates (or fractions thereof)have been used to supplement protein-free media.

4. Fed-Batch Cultivation

Fed-batch cultivation of cells is typically used for industrialproduction of biomolecules, such as proteins, to increase cellconcentration and to extend culture lifetime for a high productconcentration and volumetric productivity. Fed-batch cultures involvethe controlled addition of one or more nutrients, in the form of feedswhich may contain nutrients that are quickly utilized by cells such asglucose, amino acids to a basal medium. The nutrient(s) help to controlthe growth of the cell culture by attempting to prevent nutrientdepletion and byproduct accumulation, important parameters, such as pH,osmolality and CO₂ concentration, within levels that promote cell growthor minimize cell death for optimal product expression. See Cell CultureTechnology for Pharmaceutical and Cell-Based Therapies, 349-386(Sadettin Ozturk and Wei-Shou Hu eds., Taylor and Francis Group 2006).Even then, fed batch cultures often result in high concentrations ofinhibitory metabolites and high osmolalities that eventually areincompatible with cell viability. In fed-batch cultivation, cells aretypically grown up to a certain time point in batch mode using a basalmedium. Subsequently, a medium supplement (concentrated feed) comprisingconcentrated solutions of a single or multiple nutrients is added toprovide nutrients, while minimizing volume increase or culture dilution.When the medium supplement is added to basal medium, it improves cellculture, as exhibited, for example, by more rapid cell growth, decreaseddoubling time, higher achievable density of cells, or higher productionor yield of biomolecule, such as protein, e.g., antibody or otherproteins of therapeutic interest.

5. Basal Media

A basal medium is typically used for maintenance of a cell culture, andcan comprise a number of ingredients, including amino acids, vitamins,organic and inorganic salts, sugars and other components, eachingredient being present in an amount which supports the cultivation ofthe cell in vitro. Basal media useful for prokaryotic cell cultureincluding bacterial and archebacterial cultures, viral cultures, plantcell culture, insect cell culture, mammalian cell culture can be usedwith the small peptide Examples of basal media include Eagle's basalmedium (BME), Eagle's minimal essential medium (EMEM), Dulbecco'smodification of Eagle's medium (DMEM), Glasgow's modification of Eagle'smedium (GMEM), Joklik's modified Eagle's medium, Alpha modified Eagle'smedium, Roswell Park Memorial Institute (RPMI) medium, Fischer's medium,Leibovitz L-15 medium, Trowell's T-8 medium, Williams' medium E,Biggers' medium, Connaught Medical Research Laboratories (CMRL) 1066medium, Ham's F10 medium, Ham's F12 medium, Iscove's modified Dulbecco'smedium (IMDM), MCDB 104, MCDB 110, MCDB 153, Medium 199, NCTC 135medium, and Waymouth's medium MB 752/1. For CHO cells, preferred basalmedia include CDCHO™, CD OptiCHO™, and Dynamis™ Medium (all from LifeTechnologies, Corp., Carlsbad, Calif.). Preferred concentrated feedsupplements for CHO cells include, but are not limited to,EfficientFeed™ A+ AGT™ (Invitrogen Cat. No. A25023), EfficientFeed™ B+AGT™ (Invitrogen Cat. No. A25030), Feed™ Kit A+, B+, C+ (Invitrogen Cat.No. A3315801), EfficientFeed™ C+ AGT™ (Invitrogen Cat. No. A25031, LifeTechnologies Corp., Carlsbad, Calif.).

6. Cells

The disclosure is directed to methods for culturing cells in a liquidmedium prepared according to streamlined method, wherein the cellsinclude primary epithelial cells (e.g., keratinocytes, cervicalepithelial cells, bronchial epithelial cells, tracheal epithelial cells,kidney epithelial cells and retinal epithelial cells) and establishedcell lines and their strains (e.g., CHO cells, 293 embryonic kidneycells, BHK cells, diploid cells for vaccine production including MRC-5,2BS and others, hybridomas, HeLa cervical epithelial cells and PER-C6retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells,BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3cells, Hep-2 cells, KB cells, LS180 cells, LS174T cells, NCI-H-548cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RA cells,WISH cells, BS-C-I cells, LLC-MK₂ cells, Clone M-3 cells, 1-10 cells,RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁ cells, PK(15) cells, GH₁cells, GH₃ cells, L2 cells, LLC-RC 256 cells, MH₁C₁ cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, or derivatives thereof),fibroblast cells from any tissue or organ (including but not limited toheart, liver, kidney, colon, intestines, esophagus, stomach, neuraltissue (brain, spinal cord), lung, vascular tissue (artery, vein,capillary), lymphoid tissue (lymph gland, adenoid, tonsil, bone marrow,and blood), spleen, and fibroblast and fibroblast-like cell lines (e.g.,CHO cells, TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells,citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells, CV-1 cells,COS-1 cells, COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells,BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALBcells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells, HSDM₁C₃ cells,KLN₂O₅ cells, McCoy cells, Mouse L cells, Strain 2071 (Mouse L) cells,L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells, NCTC clones 2472 and2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells,C_(II) cells, and Jensen cells, Sp2/0, NS0, NS1 cells or derivativesthereof), a stem cell, an iPSC cells, etc. Any of the above cells can beengineered to expresses: a recombinant product, a protein or arecombinant protein, a fusion product or protein, an antibody or anantibody fragment, a fusion or modified antibody, a virus or a viralproduct or component, a VLP (viral like particle), a cell, a nucleicacid, a lipid, a hormone, a steroid, or a glycoprotein.

Therefore, the disclosure is also directed to methods for making abiological product in a liquid medium prepared according to streamlinedmethod, wherein the biological product is a recombinant product, aprotein or a recombinant protein, a fusion product or protein, anantibody or an antibody fragment, a fusion or modified antibody, a virusor a viral product or component, a VLP (viral like particle), a cell, anucleic acid, a lipid, a hormone, a steroid, or a glycoprotein.

Example 1: Making Liquid Medium with Minimal Addition of Acid/Base

The current common liquid manufacturing process utilizes large volumesof acids and bases to solubilize categorical groups of components suchas amino acids. This process has several disadvantages. Excessive acidand base usage are associated with lot-to-lot variability due to metalcontaminants and potential modification or damage to media components.The bioproduction industry has voiced concern over the lack ofconsistency and control of metal concentrations in cell culture mediaand potential cell toxicity due to metals. In addition, regulatoryagencies are requiring lower levels of heavy metals in products forbetter disposal and environmental concerns.

In order to reduce this variability, a novel manufacturing approach wasdeveloped which relies on groupings of components, for e g, amino acids,optimizing component forms, and optimizing orders of addition toconsiderably reduce the amount of acid and base previously utilized tosolubilize media components. In an example, the liquid manufacturingprocess for the culture medium CDCHO™ (from Life Technologies, Corp.,Carlsbad, Calif.; see Table 2 below) was modified by incorporating themost soluble forms of components and by optimizing the grouping ofcomponents and the order of addition based on: 1) pH, 2) concentrationand 3) component sensitivity or reactivity. The new process resulted ina 22-fold reduction in the use of 5N acids and bases, as well as thecomplete elimination of 12N concentrated acid. This approachdemonstrated a reduction in acid/base associated contaminant metals witha 3-fold reduction in chromium and a 2-fold in nickel to the finalformulation.

The new method also streamlined and simplified the manufacturing processby reducing the number of side solutions, pH adjustments and formulationprocess time, which resulted in significant manufacturing costreductions and increased safety. In particular, with CDCHO™, the numberof side solutions was reduced by 27% and the number of acid/baseadditions and pH adjustments decreased by 33%; which overall, resultedin a reduction to formulation time of 43%. This method also enabled moreconsistency between lot-to-lot metal ion concentration, which maycontribute to less variability in performance. In fact, improvedperformance in CDCHO™ prepared by streamlined method was observed overCDCHO™ media prepared by the concentrate technology, as can be seen inFIG. 6.

Methods

Initially, components were assessed for optimal solubility at desiredpH.

Components were then grouped into component categories, for e.g., aminoacids, vitamins, salts, sugars, trace metals, buffers, and a groupcomprising sensitive or reactive components. The sensitive or reactivegroup included these components, but are not limited to these only,namely: polyamines, reactive magnesium chloride, choline chloride,hygroscopic glucose and pluronic. Some of these components may exhibitpoor solubility or pH drift due to long mixing times.

Components were assessed for weighability at a final g/L concentration(currently 1 mg/L). Components below the threshold needed concentratedside solutions that were freshly prepared.

Components were assessed for upper solubility limits based on the MerckIndex, or based on common knowledge known in the literature, orstandardized internally using methods routine in the art. Componentsabove the limits needed to be solubilized with minimal acid or baseprior to main tank addition.

Components that require a chemical reaction (for example, iron chelates)need to be prepared freshly separately, to manage the reactionrequirements (for e.g., to manage pH, process, timeline).

Each group of components were re-examined to determine the optimal pHgrouping, and components with similar pH were separated into acidic,basic and neutral sub-groups. This is especially critical for aminoacids. An exemplary classification of amino acids is shown in Table 2.Bench top confirmation of the groupings is highly recommended beforepreparation of the final formulation.

The order of addition of components into the final formulation tank wasbased on: 1) pH and 2) concentration and 3) component sensitivity orreactivity. These were standardized/confirmed with a benchtop pilot runat 20 liter minimum scale, with an additional testing at 100 liter.

The CD media components used in Example 1 is shown in Table 2:

TABLE 2 CD Media Components Particularly Preferred PreferredConcentration Range Embodiment Embodiment Ingredient (g/L) (g/L) About(g/L) L-arginine 0.1000-0.7200 0.4 0.36192 L-asparagine•H₂O0.1000-1.8000 0.9 0.90480 L-aspartic acid 0.0100-0.3600 0.7 0.18096L-glutamic acid 0.1000-0.6000 0.3 0.27144 L-histidine 0.0600-0.3600 0.20.18096 hydroxy-L-proline 0.0040-0.3600 0.2 0.18096 L-isoleucine0.1000-0.7200 0.4 0.36192 L-leucine 0.1000-1.1000 0.5 0.54288L-lysine•HCl 0.2000-1.1000 0.5 0.54288 L-methionine 0.0500-0.2400 0.10.12667 L-phenylalanine 0.0900-0.4200 0.2 0.21715 L-proline0.0500-1.1000 0.5 0.54288 L-serine 0.1000-1.1000 0.5 0.54288 L-threonine0.1000-0.7200 0.4 0.36192 L-tryptophan 0.0200-0.4200 0.2 0.20810L-tyrosine 0.1000-03600 0.2 0.18096 L-valine 0.1000-0.7200 0.4 0.36192L-cystine•2HCl 0.0200-0.2200 0.1 0.10496 Na₂HPO₄ (anhydrous)0.2000-2.5000 0.6 0.63336 pyridoxine•HCl 0.0010-0.0072 0.004 0.00362thiamine•HCl 0.0010-0.0072 0.004 0.00362 glutathione 0.0006-0.0036 0.0020.00181 zinc sulfate•7H₂O 0.0003-0.0032 0.002 0.00156 cupricsulfate•5H₂O 0.000001-0.000009 0.000005 0.000004524 cadmium0.000004-0.000040 0.00002 0.000020629 chlolde•5H₂O cobalt chloride•6H₂O0.0000006-0.0000086 0.000004 0.000004343 stannous 0.00000001- 0.00000010.000000101 chloride•2H₂O 0.00000020 manganous 0.00000001- 0.00000020.000000152 sulfate•H₂O 0.00000030 nickel sulfate•6H₂O 0.00000005-0.0000001 0.000000118 00000024 sodium metavanadate 0.0000003-0000012  0.0000006 0.000000561 ammonium 0.00000300- 0.000005 0.000005429molybdate•4H₂O 0.0000110 barium acetate 0.00000065- 0.000001 0.0000011760.00000240 potassium bromide 0.00000003- 0.00000005 0.0000000540.00000011 potassium iodide 0.000000045- 0.00000008 0.0000000810.00000016 chromium sulfate 0.000000165- 0.0000003 0.0000002990.00000060 sodium fluoride 0.00000105- 0.000002 0.000001810 0.00000360silver nitrate 0.000000045- 0.00000008 0.000000081 0.00000016 rubidiumchloride 0.00000035- 0.0000006 0.000000633 0.0000013 zirconyl chloride0.0000008-0.0000029 0.000001 0.000001448 aluminum chloride0.0000003-0.0000011 0.0000005 0.000000543 germanium dioxide 0.000000135-0.0000002 0.000000244 0.00000049 titanium tetrachloride 0.00000025-0.0000005 0.000000452 0.0000009 sodium metasilicate 0.00005-0.000950.0005 0.000452400 MgCL₂ (anhydrous) 0.0100-0.1400 0.07 0.06985D-Calcium 0.0020-0.0060 0.004 0.00362 pantothenate Calcium nitrate•4H₂O0.01800-0.3600  0.09 0.09048 KCl 0.3340-1.4500 0.7 0.72384 Ascorbicacid, 0.00199-0.040   0.02 0.01991 Mg salt phosphate Pluronic F68, 5.0mL-40.0 mL/L 18 18.096 10% Solution (0.5-4.0 g/L) (2 (1.8096 Na₂HPO₄(anhydrous) 0.018-0.360 0.09 0.09048 D-glucose 1.000-12.60 6 6.33360folic acid  0.002-0.0072 0.004 0.00362 riboflavin  0.0002-0.00072 0.00040.000362 biotin 0.000575-0.00360  0.002 0.00181 choline chloride0.0280-0.1810 0.09 0.09048 niacinamide  0.0003-0.00724 0.004 0.00362i-inositol 0.0260-0.127  0.06 0.06334 sodium pyruvate 0.070-0.400 0.20.19906 vitamin B-12 0.0005-0.0018 0.0009 0.00090 β-mercaptoethanol0.00014-0.00282 0.001 0.00141 para-aminobenzoic  0.0010-0.00362 0.0020.00181 acid β-glycerophosphate 0.090-1.800 0.9 0.90480 sodium selenite0.00000157-0.000032  0.00002 0.0000157 ethanolamine•HCl 0.0075-0.02800.01 0.01357 spermine 0.0009-0.0181 0.009 0.00905 putrescine•2HCl0.00012-0.00110 0.0005 0.000543 monothioglycerol 0.0100-0.0362 0.020.01810 NaHCO₃ 1-4 2 2.22

The iron chelate compound is added as indicated in FIG. 1 to the 1×medium prior to filter sterilization.

An Exemplary Method for making liquid media using the streamlinedmethod: any exemplary liquid formulation as shown in Tables 2 above, andTables 3-5 below can be used in the streamlined method

1) Modify and convert an amino acid's free base form/salt form to targeta particular pH according to the components in the formulation.2) Modify amino acid group into multiple sub-groups according to pH withacidic, basic and neutral groupings with order of addition optimizedbased on theoretical solubilities, pH and bench top testing. Anexemplary order of amino group addition to the formulation tank wasdetermined to be as follows: 1) acidic, 2) basic and 3) neutral.3) Modify and convert equimolar phosphate form from all dibasic sodiumphosphate to a combination of monobasic and dibasic forms to achieve adesired final target pH.4) Optionally, convert equimolar spermine form to sperminehydrochloride.5) Unnecessary side solutions were removed and incorporated as mainformulation tank direct additions (meaning, instead of solutions,components were directly added in their native forms).6) Modify current multiple trace element side solution additions to themain formulation tank with creation and addition of one, complete, traceelement solution (combine trace element solutions).7) Modify solubilization process for folic acid (above solubility limit)from freshly prepared pH solution to minimal sodium hydroxidesolubilized side solution.8) Incorporated new, freshly prepared iron chelation solution bestpractice of targeting acid addition based on iron and EDTAconcentration.9) Modify vitamin group to remove choline chloride (hygroscopic) into aseparate group.10) Modify glucose into its own new group (potential reactivity and mostfrequent custom modification).11) Created a higher concentration salts group with approximately 85% offinal theoretical sodium chloride. (Previously only osmolalityadjustment addition.)

12) Created ˜7.5% pluronic side solution to minimize foaming in tank andreduce mix time to solubilize. (Previously direct addition at end).

The ingredients listed in the following Tables 3-5 below, can be admixedtogether according to the methods described above, to form a completeliquid culture medium prepared by the streamlined method. These completemedia are suitable for use in the culture of a variety of mammaliancells, for e.g., 293 cells. 293 cells are also referred to as HEK 293cells. Based on the information presented in Tables 3-5, and theknowledge possessed by those of ordinary skill in the art, one canobtain operative streamlined liquid media formulations without undueexperimentation.

TABLE 3 Complete Medium Preferred Ranges of Ingredients Gram(s)/L(unless Gram(s)/L noted) (unless noted) COMPONENT about: COMPONENTabout: L-Arginine HCl 0.05-2.50 Ammonium Meta 0.0000001- Vanadate0.000003 L-Asparagine 0.005-0.05  Manganous 0.00000001- Chloride 4H₂O0.0000005 L-Aspartic Acid 0.03-0.8  Pyridoxine HCl 0.0002-0.005 L-Cysteine HCl 0.03-0.8  Thiamine HCl 0.0002-0.006  H₂O L-Glutamic Acid0.002-0.05  Ferric Nitrate 0.0002-0.004  9H₂O L-Histidine HCl 0.01-0.3 Magnesium 0.005-0.1   H₂O Sulfate L-Isoleucine 0.04-1   Zinc Sulfate0.00008-0.002   L-Leucine 0.06-1.5  Zinc Chloride 0.00005-0.001  L-Lysine HCl 0.05-1.25 Ascorbic Acid 0.00000005- 0.0000003 L-Methionine0.02-0.6  D-Calcium 0.0002-0.006  Pantothenate L-Phenylalanine 0.02-0.5 Calcium Chloride 0.002-0.06  L-Serine 0.07-2   Magnesium 0.02-0.4 Chloride L-Threonine 0.02-0.6  Potassium 0.06-1.2  Chloride L-Tryptophan0.008-0.2   Sodium Selenite 0.0000000001- 0.00000003 L-Valine .03-1  Vitamin B12 0.00002-0.005   L-Tyrosine 2Na 2 0.02-0.5  Choline Chloride0.003-0.07  H₂O L-Glutamine 0.1-3   i-Inositol 0.004-0.09  (recommendedaddition, formulation does not contain) D-Glucose  1-25 Niacinamide0.0002-0.006  (Dextrose) Lipoic Acid 0.0004-0.01   Ethanolamine HCl0.001-0.3   Linoleic Acid 0.00001- Putrescine 2HCl 0.00003-0.008  0.0003 Folic Acid 0.0001-003   Sodium Pyruvate 0.02-0.6  HEPES 0.5-15 Riboflavin 0.00004-0.001   2- 0.0006-0.02  Biotin 0.00002-0.0005 Hydroxypyridine- n-oxide Pluronic F-68* 0.06-2   Sodium 0.5-2  Bicarbonate Sodium Phosphate 0.03-0.6  Sodium 0.6-16 *** Monobasic H₂OChloride *** *** Added to adjust osmolarity. Quantity will vary somewhatbetween formulations. NaCL is added to adjust osmo from ~160 to a valuewithin 275+/−5 mOsmo

TABLE 4 Exemplary Medium Gram(s)/L (unless Gram(s)/L noted) (unlessnoted) COMPONENT about: COMPONENT about: L-Arginine HCl 0.4 AmmoniumMeta 0.0000006 Vanadate L-Asparagine 0.03 Manganous 0.0000001 Chloride4H₂O L-Aspartic Acid 0.2 Pyridoxine HCl 0.001 L-Cysteine HCl 0.2Thiamine HCl 0.001 H₂O L-Glutamic Acid 0.01 Ferric Nitrate 9H₂O 0.0008L-Histidine HCl 0.06 Magnesium Sulfate 0.02 H₂O L-Isoleucine 0.2 ZincSulfate 0.0004 L-Leucine 0.3 Zinc Chloride 0.0003 L-Lysine HCl 0.2Ascorbic Acid 0.0000003 L-Methionine 0.1 D-Calcium 0.001 PantothenateL-Phenylalanine 0.1 Calcium Chloride 0.01 L-Serine 0.4 Magnesium 0.08Chloride L-Threonine 0.1 Potassium Chloride 0.3 L-Tryptophan 0.04 SodiumSelenite 0.000000007 L-Valine 0.2 Vitamin B₁₂ 0.001 L-Tyrosine 2Na 2 0.1Choline Chloride 0.01 H₂O L-Glutamine 0.6 i-Inositol 0.02 (recommendedaddition, formulation does not contain) D-Glucose 5 Niacinamide 0.001(Dextrose) Lipoic Acid 0.002 Ethanolamine HCl 0.005 Linoleic Acid0.00006 Putrescine 2HCl 0.0002 Folic Acid 0.0005 Sodium Pyruvate 0.1HEPES 3.0 Riboflavin 0.0002 2- 0.003 Biotin 0.0001 Hydroxypyridine-n-oxide Pluronic F-68* 0.3 Sodium 2.0 Bicarbonate Sodium Phosphate 0.1Sodium 3.0 *** Monobasic H₂O Chloride *** *** Added to adjustosmolarity. Quantity will vary somewhat between formulations. NaCL isadded to adjust osmo from ~160 to a value within 275+/−5 mOsmo

TABLE 5 Well Defined Medium Gram(s)/L Gram(s)/L (unless (unless noted)noted) COMPONENT about: COMPONENT about: L-Arginine HCl 0.43 AmmoniumMeta 0.0000006 Vanadate L-Asparagine 0.0264 Manganous Chloride 0.00000014H₂O L-Aspartic 0.15 Pyridoxine HCl 0.00103 Acid L-Cysteine HCl 0.15Thiamine HCl 0.00112 H₂O L-Glutamic 0.01 Ferric Nitrate 9H₂O 0.00081Acid L-Histidine 0.06 Magnesium Sulfate 0.0241 HCl H₂O L-Isoleucine 0.2Zinc Sulfate 0.000375 L-Leucine 0.3 Zinc Chloride 0.00025 L-Lysine HCl0.25 Ascorbic Acid 0.00000025 L-Methionine 0.115 D-Calcium Pantothenate0.00119 L- 0.1 Calcium Chloride 0.0111 Phenylalanine L-Serine 0.388Magnesium Chloride 0.0762 L-Threonine 0.12 Potassium Chloride 0.2763L-Tryptophan 0.04 Sodium Selenite 0.0000000067 L-Valine 0.19 Vitamin B₁₂0.00103 L-Tyrosine 2Na 0.1 Choline Chloride 0.014 2 H₂O L-Glutamine0.584 i-Inositol 0.018 (recommended addition, formulation does notcontain) D-Glucose 5 Niacinamide 0.00122 (Dextrose) Lipoic Acid 0.002Ethanolamine HCl 0.005 Linoleic Acid 0.00006 Putrescine 2HCl 0.00016Folic Acid 0.0005 Sodium Pyruvate 0.11 HEPES 2.98 Riboflavin 0.00022 2-0.003 Biotin 0.000097 Hydroxypyridine- n-oxide Pluronic F-68* 0.3 SodiumBicarbonate 2.4 Sodium 0.125 Sodium Chloride *** 3.126 *** PhosphateMonobasic H₂O *** Added to adjust osmolarity. Quantity will varysomewhat between formulations. NaCL is added to adjust osmo from ~160 toa value within 275+/ −5 mOsmo

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A method of making a liquid medium comprising: i)grouping media components into groups based on their pH, solubility andconcentration, ii) converting an amino acid's free base form/salt formto target a particular pH according to the components in theformulation; iii) performing addition of the groups in an order thatpermits optimal solubilization of said group; wherein the resultingliquid medium requires lesser acid and/or base addition to solubilizethe groups compared to a medium that is not prepared using steps i) ii)and iii) above.
 2. The method of making a liquid medium according toclaim 1 wherein the resultant liquid medium: (a) requires lesser acidand/or base additions to solubilize the components; and/or, (b) hasreduced metal contaminants from acid and/or base additions; and/or, (c)has less lot-to-lot variability in metals, (d) requires lesserformulation time since lesser solutions need to be prepared, compared toa liquid medium prepared by another method that does not use the stepsi), ii) and iii) of claim 1 in the preparation of the liquid medium. 3.The method of making a liquid medium according to claims 1-2 wherein,the lot-to-lot variability in metals is less than up to 10%.
 4. Themethod of making a liquid medium according to claims 1-3 wherein thelot-to-lot variability of total metal concentrations is less than about0.001%, less than about 0.01%, less than about 0.1%, less than about 1%,less than about 1-2%, less than about 1-3%, less than about 1-4%, lessthan about 1-5%, less than about 1-6%, less than about 1-10%, less thanabout 1-20%, less than about 1-30%, less than about 1-40%, less thanabout 1-50%, less than about 1-60%, less than about 1-70%, %, less thanabout 1-80%, or less than about 1-90%.
 5. The method of making a liquidmedium according to claims 1-4 wherein the lot-to-lot variability forany metal concentration between various liquid lots is less than about0.001%, less than about 0.01%, less than about 0.1%, less than about 1%,less than about 2%, less than about 3%, less than about 4%, less thanabout 5%, less than about 6%, less than about 10%, less than about 15%,less than about 20%, less than about 30%, less than about 40%, less thanabout 50%, less than about 0.001-1%, less than about 0.001-5%, less thanabout 0.001-10%, less than about 1-10%, less than about 10-20%, lessthan about 20-30%, less than about 30-40%, less than about 40-50%, lessthan about 50-100%, less than about 50-90%, less than about 50-80%, lessthan about 50-70%, less than about 50-60%, and so on.
 6. The method ofmaking a liquid medium according to claims 1-5, wherein the reduction inthe volume of acids and bases used is about 10-40 fold.
 7. The method ofmaking a liquid medium according to claims 1-6, wherein the reduction inthe volume of acids and bases used is about 22 fold.
 8. The method ofmaking a liquid medium according to claims 1-7, wherein 12N concentratedacid use is eliminated in the method.
 9. The method of making a liquidmedium according to claims 1-8, wherein the reduction of the one or morecontaminant metal was about 0.0001% to 100% in the resultant liquidprepared by the method according to claim 1 compared to liquid mediumprepared by any other method.
 10. The method of making a liquid mediumaccording to claims 1-9, wherein the reduction of the one or morecontaminant metal is by about 0.0001% to 0.001%, about 0.0001% to 0.01%,about 0.0001% to 0.1%, about 0.0001% to 1%, about 0.0001% to 2%, about0.0001% to 3%, about 0.0001% to 4%, about 0.0001% to 5%, about 0.0001%to 10%, about 1%-5%, about 1%-10%, about 1%-15%, about 1%-20%, about1%-25%, about 1%-35%, about 10-20%, about 10-30%, about 10-40%, about10-50%, about 10-60%, about 10-70%, about 10-80%, about 10-90%, about10-100% in the resultant liquid prepared by the method according toclaim 1 compared to liquid medium prepared by any other method.
 11. Themethod of making a liquid medium according to claims 1-10, wherein themetals that are reduced are selected from the group consisting of: Cr,Fe, Mg, Cu, Mn, Ni, Zn, Mo, Al and Ca.
 12. The method of making a liquidmedium according to claim 11, wherein the % metal contamination isreduced by: about 40-100% for Cr, about 0.01-20% for Fe, about 0.1-15%for Cu, about 0.25-60% for Mn, about 5-100% for Ni, about 5% for Zn, andabout 15% for Mo compared to liquid medium prepared by any other method.13. A method for culturing a cell, comprising contacting the cell in aliquid cell culture medium prepared according to claim
 1. 14. The methodof claim 13, wherein the liquid cell culture medium is serum-free. 15.The method of claim 13 to 14, wherein the liquid cell culture medium isprotein-free.
 16. The method of claims 13 to 14, wherein the liquid cellculture medium has lower metal levels than a liquid medium prepared byanother method.
 17. The method of claims 13-16, wherein the cells areselected from the group consisting of: primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells), established cell lines and their strains,recombinantly engineered cells, diploid cells, hybriomas, Chinesehamster ovary (CHO), HEK293 cells, TRG-2 cells, IMR-33 cells, Don cells,GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells,Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299cells, IMR-90 cells, MRC-5 cells, WI-38 cells, WI-26 cells, MiCl₁ cells,CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, Vero cells,DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C₃H/IOTI/2 cells,HSDM₁C₃ cells, KLN₂O₅ cells, McCoy cells, Mouse L cells, Strain 2071(Mouse L) cells, L-M strain (Mouse L) cells, L-MTK⁻ (Mouse L) cells,NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indianmuntjac cells, SIRC cells, C_(II) cells, and Jensen cells, Sp2/0, NS0,NS1 cells or derivatives thereof and iPSC cells.
 18. The method ofclaims 13-17, wherein the cell expresses: a recombinant product, aprotein or a recombinant protein, a fusion product or protein, anantibody or an antibody fragment, a fusion or modified antibody, a virusor a viral product or component, a VLP (viral like particle), a cell, anucleic acid, a lipid, a hormone, a steroid, or a glycoprotein.
 19. Amethod for making a biological product in a liquid medium preparedaccording to claim 3 13-18, wherein the biological product is arecombinant product, a protein or a recombinant protein, a fusionproduct or protein, an antibody or an antibody fragment, a fusion ormodified antibody, a virus or a viral product or component, a VLP (virallike particle), a cell, a nucleic acid, a lipid, a hormone, a steroid,or a glycoprotein.
 20. A liquid cell culture medium comprising reducedmetal contaminants and/or showing reduced lot-to-lot variability inmetals compared to a liquid medium prepared using another method whereinit uses acid and/or base additions to solubilize its components.
 21. Aliquid cell culture medium prepared according to the methods of claims 1to
 12. 22. A kit or a combination comprising: (i) a liquid cell culturemedium prepared according to the method of claims 1-12, (ii) cells, andoptionally, (iii) a cell culture supplement or additive.